Vaccines containing recombinant proteins require an adjuvant to elicit an immune response. (Callahan et al., 1991, The importance of surface charge in the optimization of antigen-adjuvant interactions, Pharm. Res. 8(7):851-858). Aluminum-salt adjuvants are currently the most widely used adjuvant for general use in humans.
The mechanisms of action of aluminum-salt adjuvants are poorly understood, but likely due to several different mechanisms. (Lindblad 2004. “Aluminium compounds for use in vaccines” Immunol. Cell. Biol. 82(5):497-505; Gupta and Siber, 1995, Adjuvants for Human Vaccines—Current Status, Problems and Future-Prospects. Vaccine 13(14):1263-1276; Gupta and Rost, 2000, Aluminum Compounds as Vaccine Adjuvants, In O'Hagan D, editor Vaccine Adjuvants: Preparation Methods and Research Protocols, ed., Totowa, N.J.: Humana Press Inc. p 65-89; Cox and Coulter, 1997, Adjuvants—a classification and review of their modes of action, Vaccine 15(3):248-256).
Common proposed mechanisms are that the adjuvant acts as a depot at the site of injection, wherein the antigen is slowly released after administration. (Cox and Coulter, 1997). Another proposed mechanism is that the adjuvant aids in delivery of the antigen to antigen-presenting cells (Lindblad 2004). A further proposed mechanism is that the adjuvant serves as an immunostimulator and elicits Th2 cytokines (Grun and Maurer 1989, Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses. Cell Immunol 121(1):134-145). Yet another proposed mechanism is that the adjuvant destabilizes protein antigens on the surface of the adjuvant making them more susceptible to proteolytic degradation (Jones et al., 2005, Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens. J Biol Chem 280(14):13406-13414; and That et al., 2004. “Antigen stability controls antigen presentation” J. Biol. Chem. 279(48):50257-50266).
Vaccines based on recombinant protein antigens must be formulated with an adjuvant for maximum potency. (Singh and O'Hagan 1999, Advances in vaccine adjuvants, Nat Biotechnol 17(11): 1075-81; and O'Hagan et al., 2001, Recent developments in adjuvants for vaccines against infectious diseases, Biomol Eng 18(3): 69-85). The only adjuvants currently appearing in FDA-approved vaccines are the aluminum salt adjuvants, aluminum hydroxide and aluminum phosphate. It has been suggested that to provide adequate immunogenicity, antigens must be adsorbed on the surface of the adjuvant. (Gupta et al., 1995, Adjuvant Properties of Aluminum and Calcium Compounds. Pharmaceutical Biotechnology. 6: 229-248; and White and Hem, 2000, Characterization of aluminium-containing adjuvants, Dev Biol (Basel) 103: 217-28). This adsorption is typically facilitated through electrostatic interactions between the antigen and adjuvant, and the formulation pH is usually chosen so that the antigen and adjuvant are oppositely charged (Callahan et al. 1991). The surface charge on the adjuvant also can be modified by surface exchange reactions with buffer salts such as phosphate, succinate, and citrate (Hem and White, 1984, Characterization of aluminum hydroxide for use as an adjuvant in parenteral vaccines. J Parenter Sci Technol, 38(1): p. 2-10; Chang et al., 1997, Role of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant. PDA J Pharm Sci Technol, 51(1): p. 25-9; and Rinella et al., 1996, Treatment of aluminium hydroxide adjuvant to optimize the adsorption of basic proteins. Vaccine, 14(4): p. 298-300.)
Although the mechanism of action is not fully understood, it is likely that surface area, surface charge, and morphology of the adjuvant are important factors dictating the immune response to antigens adsorbed onto these adjuvants (Hem and White 1984). It is generally theorized that the smaller the particle size of the vaccine adjuvant, the more immunogenic the vaccine preparation (Maa et al., 2003. Stabilization of alum-adjuvanted vaccine dry powder formulations: mechanism and application. J Pharm Sci 92(2):319-332., Diminsky et al., 1999. Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles. Vaccine 18(1-2):3-17).
Lyophilization (freeze drying) is a process frequently utilized to improve long term stability of various protein preparations. However, when vaccines formulated with aluminum-salt adjuvants are processed in an attempt to improve stability through freezing and lyophilization, a loss of potency is often reported. Previous studies have suggested that a freeze-dried vaccine product containing an adjuvant cannot be produced due to aggregation of the adjuvant particles. (Diminsky et al., 1999; Maa et al., 2003).
A number of theories were previously set forth to explain possible mechanisms responsible for the loss of potency following lyophilization of vaccines formulated with aluminum-salt adjuvants. For example, the aggregation of aluminum hydroxycarbonate and magnesium hydroxide gels after freezing and thawing has been attributed to ice crystal formation which forces particles together, resulting in irreversible aggregation. (Zapata et al., 1984, Mechanism of freeze-thaw instability of aluminum hydroxycarbonate and magnesium hydroxide gels. J Pharm Sci 73(1):3-8). This explanation has been echoed by Maa et al., 2003 who propose that faster cooling rates result in a greater rate of ice nucleation and the formation of smaller ice crystals, which would not force alum particles into an aggregate. Nygaard et al. showed that the particle diameter, and thus surface area and number of particles, and not mass or volume, is the dominant property in the immunological response of polystyrene particles in mice (Nygaard et al., 2004). Many of these proposed mechanisms have since been shown to be incorrect.
Roser et al., U.S. Pat. No. 6,890,512 disclose a method of preventing aggregation during dehydration and rehydration of particulates in suspension by adding to a particulate suspension of aluminum hydroxide at least 15% (w/v) of trehalose. However, Roser et al. does not discuss freezing rate.
The capacity of particles to increase allergic sensitization is predicted by particle number and surface area, not by particle mass. Toxicol Sci 82(2):515-524). Moorefield et al. showed that the degree of antigen internalization of adjuvant particles is inversely related to the particle size of the adjuvant aggregates (Moorefield et al., 2005. “Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro” Vaccine 23(13):1588-1595). While it is likely that the particle size is an important characteristic parameter for immunogenicity, there has yet to be a comprehensive study examining the particle size distribution (PSD) as a function of formulation and cooling rates along with other physical properties of the products produced.
The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. (See e.g., P. H. A. Sneath et al., 1986, “Clostridium,” Bergey's Manual® of Systematic Bacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins). Only a few of the approximately 100 species of Clostridium have been recognized as etiologic agents of medical and veterinary importance. However, these species are associated with very serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis. In most cases, the pathogenicity of these organisms is related to the release of powerful exotoxins or highly destructive enzymes. (Hatheway, 1990, Clin. Microbiol. Rev., 3:66-98).
The botulinum neurotoxin is one of the most poisonous substances known, and hence it has been identified as a potential threat for biological warfare (Gill 1982, Bacterial toxins: a table of lethal amounts, Microbiol Rev 46(1): 86-94; Caya 2001, Clostridium botulinum and the ophthalmologist: a review of botulism, including biological warfare ramifications of botulinum toxin, Surv Ophthalmol 46(1): 25-34). The lethal human dose is 10−9 mg/kg bodyweight for toxin in the bloodstream. Produced by the bacteria Clostridium botulinum, the neurotoxin exists as seven structurally similar but serologically different variants, identified as A through G (Oguma, et al., 1995, Structure and function of Clostridium botulinum toxins, Microbiol Immunol 39(3): 161-8; Caya 2001). There is little, if any, antibody cross-reactivity between the seven BoNT serotypes A to G. The proteins are comprised of a 100 kDa heavy chain containing binding and internalization domains, and a 50 kDa light chain comprising the catalytic domain, connected by a disulfide bond. Botulinum neurotoxin blocks nerve transmission to the muscles, resulting in flaccid paralysis. When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death. (Amon, 1986, J. Infect. Dis. 154:201-206).
Botulism disease is grouped into four types, based on the method of introduction of toxin into the bloodstream. Food-borne botulism results from ingesting improperly preserved and inadequately heated food that contains botulinal toxin. (MacDonald et al., 1986, Am. J. Epidemiol. 124:79). Wound-induced botulism results from C. botulinum penetrating traumatized tissue and producing toxin that is absorbed into the bloodstream. (Swartz, 1990 “Anaerobic Spore-Forming Bacilli: The Clostridia,” pp. 633-646, in B. D. Davis et al., (eds.), Microbiology, 4th edition, J. B. Lippincott Co.). Infectious infant botulism results from C. botulinum colonization of the infant intestine with production of toxin and its absorption into the bloodstream. It is likely that the bacterium gains entry when spores are ingested and subsequently germinate. (Amon 1986). Inhalation botulism results when the toxin is inhaled. Inhalation botulism has been reported as the result of accidental exposure in the laboratory (E. Holzer, Med. Klin. 41:1735 (1962)) and could arise if the toxin is used as an agent of biological warfare (Franz et al., in Botulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), pp. 473-476).
Different strains of Clostridium botulinum each produce antigenically distinct toxin designated by the letters A-G. Serotype A toxin has been implicated in 26% of the cases of food botulism; types B, E and F have also been implicated in a smaller percentage of the food botulism cases. (Sugiyama 1980, Clostridium botulinum neurotoxin, Microbiol. Rev. 44:419-448). Sequence variation within various C. botulinum neurotoxin serotypes are described in Smith et al. (Smith et al., Sequence variation within Botulinum Neurotoxin Serotypes impacts antibody binding and neutralization. Infection Immun. 2005, 73 (9): 5450-5457).
Currently available protein therapies against these toxins are inadequate because of limited availability, high production costs, and potential side effects (Eubanks et al., 2007, An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists, Proc. Nat. Acad. Sci., 104(8):2602-2607) Immunization of subjects with toxin preparations has been done in an attempt to induce immunity against botulinal toxins. A C. botulinum vaccine comprising chemically inactivated (i.e., formaldehyde-treated) type A, B, C, D and E toxin is commercially available for human usage. However, this vaccine preparation has several disadvantages. First, the efficacy of this vaccine is variable (in particular, only 78% of recipients produce protective levels of anti-type B antibodies following administration of the primary series). Second, immunization is painful (deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% of recipients upon initial injection; this number rises to approximately 11% of individuals who receive booster injections) (Informational Brochure for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control). Third, preparation of the inactivated vaccine is dangerous as active toxin must be handled by laboratory workers.
Recombinant proteins and peptides derived from the Clostridium botulinum neurotoxin are useful as immunogens for the production of vaccine compositions of the present disclosure. For example, several such recombinant protein sequences, recombinant production techniques and immunological assays are described in U.S. Pat. No. 5,919,665, which is incorporated herein by reference. Recombinant protein antigens for the seven serotypes have been created as part of the development of a heptavalent vaccine against the neurotoxins (Smith 1998, Development of recombinant vaccines for botulinum neurotoxin, Toxicon 36(11): 1539-48; and Smith et al., 2004, Roads from vaccines to therapies, Mov Disord 19 Suppl 8: S48-52). These protein antigens (identified as rBoNTA(Hc)-rBoNTG(Hc)) consist of 50 kDa portions of the C-terminal domain of the heavy chains and have no neurotoxin activity (DePaz et al., 2005, Formulation of Botulinum Neurotoxin Heavy Chain Fragments for Vaccine Development: Mechanisms of Adsorption to an Aluminum-Containing Adjuvant, Vaccine 23: 4029-4035).
Paralysis-inducing neurotoxins produced by the bacterium Clostridium botulinum are highly toxic proteins to humans and are classified as category A bioagents by the U.S. government. There is a continuing need to improve vaccine safety and stability without the loss of vaccine immunogenicity. Due to potential bioterrorism threats, there is also an increased need for safe, stable and effective vaccine compositions for administration to those at risk of exposure to C. botulinum neurotoxins.
One way to accomplish these goals is to develop methods of production of stable, immunologically-active freeze dried vaccine preparations which may incorporate recombinant antigens.